This invention relates to controlled molecular weight reduction of polymers; more particularly, it relates to a pressure homogenization method which is particularly suitable for weight reduction of polysaccharides.
It has now surprisingly been found that controlled, reproducible molecular weight regulation of polymers, and in particular polysaccharides, such as hyaluronic acid (HA), carboxymethyl cellulose (CMC) and guar gum (GG), may be achieved using the technique of pressure homogenization. The diverse nature of these polysaccharides suggests that the present method may be widely applicable more generally to all polymers. However, as the primary focus of this invention is HA, the present invention will be described with particular reference thereto.
HA is a linear long-chain polysaccharide comprising repeating D-glucoronate and N-acetyl glucosamine disaccharide units. It is species non-specific and may be obtained, for example, either by extraction from animal tissues, e.g. rooster combs and umbilical cords, (Klein, J., & Meyer, F. A., 1983, Biochem. & Biophys. Res. Comm., 755, 400-411), or by removal of HA capsular material form bacterial species, e.g. Streptococcus , (Van Brunt, J., 1986, Biotechnology, 4, 780-782). HA from such sources exists as a mixture of different molecular weight species and the overall weight is expressed as a weight average.
HA has a variety of therapeutic applications, such as opthalmic surgery and post-operative adhesion prevention, as well as having potential uses in a number of other areas. The key to the uses of HA is its hydrodynamic properties (Van Brun, loc cit), producing highly viscous solutions at low concentrations. The viscosity of an HA solution is primarily dependent on the molecular weight, as well as the concentration of the HA.
Viscosity properties may be altered by dilution of the material, but in many applications this is not acceptable. Proposed strategies to produce HA having a variety of molecular weights include the selection of bacterial mutants capable of producing the desired HA molecular weight range or the alteration of the physiological conditions during growth of a bacterium to enhance the production of a particular molecular weight range. However, neither of these techniques could provide both the range and diversity of molecular weight species required for specific applications.
Alternatively, reduction of high molecular weight HA to moieties of lower molecular weight may be achieved by enzymatic, chemical or physical means. Enzymatic methods of degrading HA are known, (Hamai, A., et al, 1989, Agric. Biol. Chem., 58(8), 2163-2168), but are relatively uncontrollable and tend to broaden the HA molecular weight distribution, therefore making the material unsuitable for certain applications where highly defined molecular weight ranges are required. Chemical methods (Harris, M. J., et al, 1972, JACS, 94, 7570-7572), suffer similar problems and, moreover may result in residual concentrations of the reacting chemicals remaining in a therapeutic product. Fractionation of HA into defined molecular weight species is feasible (Armand, G., & Reyes, M., 1983, Biochem. & Biophys. Res. Comm., 112(1), 168-175), but is a complex operation and so is not easily controllable in large scale manufacture.
With regard to physical means, it has long been a generally held belief that long-chain polysaccharides are shear-sensitive. Recently, the use of a high shear disc stack centrifuge for cell removal from fermentation broth due to the process volume was attempted and, surprisingly, little or no reduction in overall molecular weight of the HA was observed. This suggested that the polysaccharide is less shear-sensitive than previously thought. Given the existing requirements for HA of various molecular weights, several possibilities had been considered. Pressure homogenization had not previously been considered due to the high shear generated, but, given the surprising observation that HA is less shear-sensitive than expected, pressure homogenization has now been investigated as a means for the controlled molecular weight reduction of such polysaccharides, as well as other polymers.